Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Carbon monoxide (CO)-releasing antibody conjugates were synthesized utilizing a photoactivatable COreleasing molecule (photoCORM) and mouse monoclonal antibodies linked by a biotin-streptavidin system. Different monoclonal antibodies raised against different surface-expressed antigens that are implicated in ovarian cancer afforded a family of antibody-photoCORM conjugates (Ab-photoCORMs). In an immunosorbent/cell viability assay, Ab-photoCORMs accumulated onto ovarian cancer cells expressing the target antigens, delivering cytotoxic doses of CO in vitro. The results described here provide the first example of an "immunoCORM", a proof-of-the-concept antibody-drug conjugate that delivers a gaseous molecule as a warhead to ovarian cancer. † Electronic supplementary information (ESI) available: Synthetic scheme and calculations for CO release (Schemes S1 and S2). Spectroscopic, chromatography and mass spectrometry data ( Fig. S1-S4, S8 and S9). Myoglobin assays and cell toxicity/viability data (Fig. S5-S7 and S10-S13). Description of the experimental procedures. See Scheme 1 Live-cell, immunosorbent assay scheme utilized for assessment of the efficacy of antigen-recognition of an antibody-(photoactivated carbon monoxide-releasing molecule) conjugate (Ab-photoCORM) to deliver cytotoxic levels of carbon monoxide to ovarian cancer cells compared to a non-specific Ab-photoCORM conjugate.This journal is

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule–kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein–protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein–protein interactions. Additionally, it uncovered new protein associations, including the ELYS–MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

SUMMARYHuman cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. Here we show that the dual specificity phosphatase DUSP7 is important for regulating chromosome alignment. DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytic dead mutants, led to a marked decrease in phopho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, chemical inhibition of the MEK kinase that phosphorylates ERK2 or ERK2 itself led to chromosome alignment defects. Our results support a model where MEK phosphorylation and DUSP7 dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson’s disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson’s disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson’s disease.

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors prior to proceeding into anaphase. The SAC has a central role in ensuring the fidelity of chromosome segregation and its dysregulation has been linked to the development of human diseases like cancer. The establishment and maintenance of the SAC relies on multiple protein complexes that are intricately regulated in a spatial and temporal manner through posttranslational modifications like phosphorylation. Over the past few decades the SAC has been highly investigated and much has been learned about its protein constituents and the pathways and factors that regulate its activity. However, the spatio-temporal proximity associations of the core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2 proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 under conditions where the SAC is active in prometaphase. These five protein association maps were integrated to generate the SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many of the known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations that lend insight into the functioning of the SAC and highlighted future areas that should be investigated to generate a comprehensive understanding of the SAC.3

Human cell division is a critical process that ensures the accurate transmission of the genetic material from one mother cell to two cells. The mitotic Spindle Assembly Checkpoint (SAC) ensures the fidelity of this process by responding to errors in microtubule‐kinetochore attachment and tension, and arresting the cells in prometaphase to allow time to repair the errors before progressing into anaphase. The SAC core proteins include MAD2, MAD1, BUB1, BUBR1, and BUB3. To gain insight into the protein associations that are critical for regulating the SAC in a spatial‐temporal manner, we have employed a label‐free proximity‐based association mapping approach. We have generated BIOID2 stable cell lines for all of the core SAC proteins and the BioID2 tag alone as our control. We have verified the inducible expression of these BioID2 tagged SAC proteins and their localization to the kinetochore region at the time when the SAC is active. We have performed purifications from mitotic arrested cells and have generated proximity association networks for these SAC proteins. Although, proteomic approaches, like BioID2, have been widely used to identify novel cellular pathway components and to interrogate their functions, a common problem that we and other researchers face is what to call a “Hit” and what to call a “contaminant” in mass proteomic data, therefore we have developed a statistical proteomic pipeline in R that 1) is user friendly and 2) can be used to analyze mass proteomic data with any lab specific control and lab specific conditions to generate statistically significant results to inform on hits vs. contaminants. We have analyzed our proximity‐based purifications with this new computational approach and have identified known and novel associations. Our current and future work is focused on better understanding these identified associations by validating them, and assessing their roles in the activation and maintenance of the SAC pathway.Support or Funding Information1. NSF MCB1243645(JZT), NIH R01GM117475‐01 (JZT),2. USHHS Ruth L. Kirschtein Institutional National Research Service Award #T32 CA009056 (YAG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

During the past decade, the incorporation of mass spectrometry data to identify protein‐protein interactions and associations has become extremely popular, and presents an exciting opportunity to exploit the protein interactome. However, the experimentation often produces large amounts of proteomic data that can be hard to handle, delaying scientific discovery. It has become clear that when handling the data, cleaning the data of false positives is as important as verifying associations. To address this issue, scientists have developed databases like the Contaminant Repository for Affinity Purification (CRAPome) that provide spectral count data for a collection of controls. However, the data can be hard to interpret without a more rigorous statistical analysis. This work describes an open source proteomic pipeline that incorporates tools to identify false positives and visualize interactions/associations in a user friendly manner. Taking advantage of R, a popular statistical programming language, and Shinny apps we developed a program that aims to standardize analysis of protein interaction/association experiments. The pipeline allows scientists to submit proteomic data and easily apply suggested significance tests to clean their experiments against their own control and the CRAPome if they wish to do so. Often these types of experiments are done through a purification step and so we have extended the platform to include predictive machine learning algorithms that highlight false positives based on intrinsic data that describes a protein that might stick to the column. After analysis, is carried out we couple the platform to protein interaction network visualization tools such as Cytoscape, to create a comprehensive analysis pipeline that is user friendly. In the future we plan on extending this platform by applying neuronal networks to further predict protein‐protein interactions, adding to a fully automated pipeline, for the analysis of protein‐interaction/association experiments.Support or Funding InformationThe work was possible through USPHS National Research Service Award 5T32GM008496 and the Whitcome Pre‐doctoral Training Program and the UCLA MolecularBiology InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.