CG -SLENP: A chemical genetics strategy to selectively label existing proteins and newly synthesized proteins.

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as CG -SLENP. Using HaloTag in-frame fusion with lamin A (LA), we demonstrate that the two pools of proteins can be selectively labeled using CG -SLENP in living cells. We further employ our recently developed selective small molecule ligand LBL1 for LA to probe the potential differences between newly synthesized and existing LA. Our results show that LBL1 can differentially modulate these two pools of LA. These results indicate that the assembly states of newly synthesized LA are distinct from existing LA in living cells. The CG -SLENP method is potentially generalizable to study any cellular proteins.

Creating a Career Path for Shared Research Resources Personnel.

Shared research resources are essential to academic research. A rapidly evolving workforce within a highly competitive market is making recruitment and retention of knowledgeable and technically skilled core staff more difficult. The inability to recruit and retain staff diminishes the resource's overall ability to provide services, which in turn affects academic research quality. Research institutions need to recognize that the roles and skills of shared research resource staff are distinguishable from those of research staff in funded investigator laboratories, and in doing so, develop a career path for shared research resource staff that will help these facilities recruit, train, and retain them. This brief focuses on the creation of a standardized career track for shared research resource staff: a career path of at least 3 to 5 tiered positions with task outlines that can be tailored to positions needed in any shared research resource. Salaries will vary for individuals within each position classification based on experience, mastered competencies, and time within the shared research resource. Besides characterizing basic task differences between shared research resource staff and other research personnel, the most compelling reason for having a well-delineated career path for shared research resource staff is to establish fairness, equity, and true opportunity in a supportive working environment, where shared research resource staff are motivated by developing a marketable skill set, gaining professional self-confidence, and earning a meaningful salary. Presented here is a case study from Oregon Health & Science University of the creation of a career path for shared research resource staff.

A clickable photoaffinity probe of betulinic acid identifies tropomyosin as a target.

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge, unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid.

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.