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Bisphenol A, an endocrine-disrupting compound, is widely used in the industrial production of plastic products. Despite increasing concerns about its harmful effects on human health, animals, and the environment, the use of BPA has been banned only in infant products, and its effects on cellular processes are not fully understood. To investigate the impact of BPA on eukaryotic cells, we analyzed the proteome changes of wild-type and PDR5-deleted S. cerevisiae strains exposed to different doses of BPA using sample multiplexing-based proteomics. We found that the ABC multidrug transporter Pdr5 plays an important role in protecting yeast cells from BPA toxicity, with its absence significantly sensitizing cells to BPA. BPA inhibited yeast growth in a dose-dependent manner, with a more pronounced effect in PDR5-deleted cells. Proteomic analysis revealed that BPA induces widespread dose-dependent changes in protein abundance, including the upregulation of metabolic pathways such as arginine biosynthesis and the downregulation of mitochondrial proteins. Additionally, we observed markers of cellular stress induced by BPA by identifying multiple stress-induced proteins that were upregulated by this compound. As cellular processes affected by BPA have been shown to be evolutionarily conserved, these insights can advance our understanding of BPA’s cellular impact and its broader effects on human health.

The Ubiquitin–Proteasome System (UPS) governs numerous cellular processes by modulating protein stability and activity via the conjugation of the small protein ubiquitin, either as a single molecule or as linkages with distinct functions. Dysregulation of the UPS has been associated with many diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Ubiquitin-conjugating enzymes (E2s) are important players of the UPS that work together with ubiquitin ligases (E3s) to promote substrate ubiquitylation. In this study, we conduct a comparative proteome-wide abundance profiling of S. cerevisiae cells during the exponential growth phase with and without heat shock treatment. We focus on cells with deletions of the two highly homologous E2s, UBC4 or UBC5, and use isobaric tag-based quantitative mass spectrometry to elucidate differences and similarities in their proteomic profiles. Our analysis revealed that the deletion of Ubc4 has a stronger effect on the proteome compared to the deletion of Ubc5, particularly in exponentially growing cells. In contrast, the effect on the proteome of deleting Ubc5 becomes evident only after heat shock, and even then, it remains minor compared to Ubc4. Furthermore, we identified proteins increasing in the absence of each enzyme, which may represent candidate substrates, potentially contributing to a better understanding of their cellular role.

The yeast ATPase Cdc48 (known as p97/VCP in human cells) plays an important role in the Ubiquitin Proteasome System. VCP is essential for cancer cell proliferation, and its dysregulation has been implicated in several neurodegenerative diseases. Cdc48 functions by extracting ubiquitylated proteins from membranes, protein complexes and chromatin by often facilitating their proteasomal degradation. Specific adaptors or cofactors, primarily belonging to the UBX domain-containing protein family (which has seven members in Saccharomyces cerevisiae) recruit Cdc48 to ubiquitylated proteins. Here, we employed sample multiplexing-based quantitative mass spectrometry to profile global protein abundance in p97 adaptor deletion strains, specifically comparing seven single deletion strains of UBX domain-containing proteins and the Cuz1 deletion strain, which belongs to the zinc finger AN1-type domain protein family. We observed that each strain showed unique sets of differentially abundant proteins compared to the wild type. Our analysis also revealed a role for Ubx3 in maintaining wild type levels of mitochondrial proteins. Overall, we identified ~1400 differentially abundant proteins in the absence of a specific Cdc48 adaptor. This unique dataset offers a valuable resource for studying the functions of these adaptors, aiming to achieve a better understanding of the cellular processes regulated by Cdc48 itself and to deepen our understanding of the Ubiquitin Proteasome System.

Human genomic DNA contains a number of diverse repetitive sequence motifs, often identified as fragile sites leading to genetic instability. Among them, expansion events occurring at triplet repeats have been extensively studied due to their association with neurological disorders, including Huntington’s disease (HD). In the case of HD, expanded CAG triplet repeats in the HTT gene are thought to cause the onset. The expansion of CAG triplet repeats is believed to be triggered by the emergence of stem-loops composed of CAG triplet repeats, while the underlying molecular mechanisms are largely unknown. Therefore, identifying proteins recruited on such stem loops would be useful to understand the molecular mechanisms leading to the genetic instability of CAG triplet repeats. We previously developed a plasmid DNA pull-down methodology that captures proteins specifically assembled on any sequence of interest using nuclear extracts. Analysis by Mass Spectrometry revealed that among the proteins specifically bound to a stem-loop composed of CAG triplet repeats, many turned out to belong to DNA repair pathways. We expect our data set to represent a useful entry point for the design of assays allowing the molecular mechanisms of genetic instability at CAG triplet repeats to be explored.

Neural regeneration and neuroprotection represent strategies for future management of neurodegenerative disorders such as Alzheimer’s disease (AD) or glaucoma. However, the complex molecular mechanisms that are involved in neuroprotection are not clearly understood. A promising candidate that maintains neuroprotective signaling networks is neuroserpin (Serpini1), a serine protease inhibitor expressed in neurons which selectively inhibits extracellular tissue-type plasminogen activator (tPA)/plasmin and plays a neuroprotective role during ischemic brain injury. Abnormal function of this protein has been implicated in several conditions including stroke, glaucoma, AD, and familial encephalopathy with neuroserpin inclusion bodies (FENIB). Here, we explore the potential biochemical roles of Serpini1 by comparing proteome changes between neuroserpin-deficient (NS−/−) and control mice, in the retina (RE), optic nerve (ON), frontal cortex (FC), visual cortex (VC), and cerebellum (CB). To achieve this, a multiple-plex quantitative proteomics approach using isobaric tandem mass tag (TMT) technology was employed followed by functional enrichment and protein–protein interaction analysis. We detected around 5000 proteins in each tissue and a pool of 6432 quantified proteins across all regions, resulting in a pool of 1235 differentially expressed proteins (DEPs). Principal component analysis and hierarchical clustering highlighted similarities and differences in the retina compared to various brain regions, as well as differentiating NS−/− proteome signatures from control samples. The visual cortex revealed the highest number of DEPs, followed by cerebellar regions. Pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation, and supramolecular fiber organization in RE, ON, FC, VC, and CB, respectively. These novel findings provide comprehensive insights into the region-specific networking of Serpini1 in the central nervous system, further characterizing its potential role as a neuroprotective agent. Data are available via ProteomeXchange with identifier PXD046873.

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein–protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

The yeast Saccharomyces cerevisiae is a powerful model system that is often used to expand our understanding of cellular processes and biological functions. Although many genetically well-characterized laboratory strains of S. cerevisiae are available, they may have different genetic backgrounds which can confound data interpretation. Here, we report a comparative whole-proteome analysis of two common laboratory yeast background strains, W303 and BY4742, in both exponential and stationary growth phases using isobaric-tag-based mass spectrometry to highlight differences in proteome complexity. We quantified over 4400 proteins, hundreds of which showed differences in abundance between strains and/or growth phases. Moreover, we used proteome-wide protein abundance to profile the mating type of the strains used in the experiment, the auxotrophic markers, and associated metabolic pathways, as well as to investigate differences in particular classes of proteins, such as the pleiotropic drug resistance (PDR) proteins. This study is a valuable resource that offers insight into mechanistic differences between two common yeast background strains and can be used as a guide to select a background that is best suited for addressing a particular biological question.

The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that, while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that, under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, implying harvesting-method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked, aspect of experimental design.

Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H2O2 at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.