azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

Endogenous anti-tumorigenic nitro-fatty acids inhibit the ubiquitin-proteasome system by directly targeting the 26S proteasome.

Nitro-fatty acids (NFAs) are endogenous lipid mediators causing a spectrum of anti-inflammatory effects by covalent modification of key proteins within inflammatory signaling pathways. Recent animal models of solid tumors have helped demonstrate their potential as anti-tumorigenic therapeutics. This study evaluated the anti-tumorigenic effects of NFAs in colon carcinoma cells and other solid and leukemic tumor cell lines. NFAs inhibited the ubiquitin-proteasome system (UPS) by directly targeting the 26S proteasome, leading to polyubiquitination and inhibition of the proteasome activities. UPS suppression induced the unfolded protein response, resulting in tumor cell death. The NFA-mediated effects were substantial, specific, and enduring, representing a unique mode of action for UPS suppression. This study provides mechanistic insights into the biological actions of NFAs as possible endogenous tumor-suppressive factors, indicating that NFAs might be key structures for designing a novel class of direct proteasome inhibitors.

SpinTip: A Simple, Robust, and Versatile Preanalytical Method for Microscale Suspension Cell Proteomics.

Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 µL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.

Identification of Non-Canonical Translation Products in C. elegans Using Tandem Mass Spectrometry.

Transcriptome and ribosome sequencing have revealed the existence of many non-canonical transcripts, mainly containing splice variants, ncRNA, sORFs and altORFs. However, identification and characterization of products that may be translated out of these remains a challenge. Addressing this, we here report on 552 non-canonical proteins and splice variants in the model organism C. elegans using tandem mass spectrometry. Aided by sequencing-based prediction, we generated a custom proteome database tailored to search for non-canonical translation products of C. elegans. Using this database, we mined available mass spectrometric resources of C. elegans, from which 51 novel, non-canonical proteins could be identified. Furthermore, we utilized diverse proteomic and peptidomic strategies to detect 40 novel non-canonical proteins in C. elegans by LC-TIMS-MS/MS, of which 6 were common with our meta-analysis of existing resources. Together, this permits us to provide a resource with detailed annotation of 467 splice variants and 85 novel proteins mapped onto UTRs, non-coding regions and alternative open reading frames of the C. elegans genome.