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Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non‐existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed inE. coliand purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

 

Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57–62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo -HLn, for “ evo lved for H eavy L antha n ides.” A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the “lanthasome.” Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo -HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo -HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery. 

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs. 

At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio‐organic molecules. However, later, metal‐based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal‐free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein‐based CEST agents, review the rationalization of enzyme‐substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein‐based CEST contrast agents.