Biology subjects

Kath, J. E.

Publications and source records attributed to Kath, J. E..

4 recordsLinked to original sources

bioRxiv2023

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

The cellular thermal shift assay (CETSA(R)) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA(R) has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA(R) in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA(R) and MSD CETSA(R) - that require less than 100 L of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA(R) platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

molecular biology↗

bioRxiv2020

Characterizing and controlling nanoscale self-assembly of suckerin-12

Structural proteins such as the "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined {beta}-sheets. Here, we report characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ~100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a non-canonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore, and the ability to change self-assembly and {beta}-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

bioengineering↗

bioRxiv2019

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

biochemistry↗

bioRxiv2017

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 {micro}g/mL for the single-subunit OST enzyme, Protein glycosylation B (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

synthetic biology↗