Chronic pain susceptibility is associated with anhedonic behavior and alterations in the accumbal ubiquitin-proteasome system.

ABSTRACT: It remains unknown why on similar acute/subacute painful conditions, pain persists in some individuals while in others it resolves. Genetic factors, mood, and functional alterations, particularly involving the mesolimbic network, seem to be key. To explore potential susceptibility or resistance factors, we screened a large population of rats with a peripheral neuropathy and we isolated a small subset (<15%) that presented high thresholds (HTs) to mechanical allodynia (reduced pain manifestation). The phenotype was sustained over 12 weeks and was associated with higher hedonic behavior when compared with low-threshold (LT) subjects. The nucleus accumbens of HT and LT animals were isolated for proteomic analysis by Sequential Window Acquisition of All Theoretical Mass Spectra. Two hundred seventy-nine proteins displayed different expression between LT and HT animals or subjects. Among several protein families, the proteasome pathway repeatedly emerged in gene ontology enrichment and KEGG analyses. Several alpha and beta 20S proteasome subunits were increased in LT animals when compared with HT animals (eg, PSMα1, PSMα2, and PSMß5). On the contrary, UBA6, an upstream ubiquitin-activating enzyme, was decreased in LT animals. Altogether these observations are consistent with an overactivation of the accumbal proteasome pathway in animals that manifest pain and depressive-like behaviors after a neuropathic injury. All the proteomic data are available through ProteomeXchange with identifier PXD022478.

Cofilin-1 Is a Mechanosensitive Regulator of Transcription.

The mechanical properties of the extracellular environment are interrogated by cells and integrated through mechanotransduction. Many cellular processes depend on actomyosin-dependent contractility, which is influenced by the microenvironment's stiffness. Here, we explored the influence of substrate stiffness on the proteome of proliferating undifferentiated human umbilical cord-matrix mesenchymal stem/stromal cells. The relative abundance of several proteins changed significantly by expanding cells on soft (∼3 kPa) or stiff substrates (GPa). Many such proteins are associated with the regulation of the actin cytoskeleton, a major player of mechanotransduction and cell physiology in response to mechanical cues. Specifically, Cofilin-1 levels were elevated in cells cultured on soft comparing with stiff substrates. Furthermore, Cofilin-1 was de-phosphorylated (active) and present in the nuclei of cells kept on soft substrates, in contrast with phosphorylated (inactive) and widespread distribution in cells on stiff. Soft substrates promoted Cofilin-1-dependent increased RNA transcription and faster RNA polymerase II-mediated transcription elongation. Cofilin-1 is part of a novel mechanism linking mechanotransduction and transcription.

Proteomics-based Predictive Model for the Early Detection of Metastasis and Recurrence in Head and Neck Cancer.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) presents high morbidity, an overall poor prognosis and survival, and a compromised quality of life of the survivors. Early tumor detection, prediction of its behavior and prognosis as well as the development of novel therapeutic strategies are urgently needed for a more successful HNSCC management. MATERIALS AND METHODS: In this study, a proteomics analysis of HNSCC tumor and non-tumor samples was performed and a model to predict the risk of recurrence and metastasis development was built. RESULTS: This predictive model presented good accuracy (>80%) and comprises as variables the tumor staging along with DHB12, HMGB3 and COBA1 proteins. Differences at the intensity levels of these proteins were correlated with the development of metastasis and recurrence as well as with patient's survival. CONCLUSION: The translation of proteomic predictive models to routine clinical practice may contribute to a more precise and individualized clinical management of the HNSCC patients, reducing recurrences and improving patients' quality of life. The capability of generalization of this proteomic model to predict the recurrence and metastases development should be evaluated and validated in other HNSCC populations.

Use of recombinant proteins as a simple and robust normalization method for untargeted proteomics screening: exhaustive performance assessment.

The label-free quantitative mass spectrometry methods, in particular, the SWATH-MS approach, have gained popularity and became a powerful technique for comparison of large datasets. In the present work, it is evaluated the use of recombinant proteins as internal standards for untargeted label-free methods. The proposed internal standard strategy reveals a similar intragroup normalization capacity when compared with the most common normalization methods, with the additional advantage of maintaining the overall proteome changes between groups (which is buffered with the use of other methods). Therefore, the proposed strategy is able to maintain a good performance even when large qualitative and quantitative differences in sample composition are observed, such as the ones induced by biological regulation (as observed in secretome and other biofluids' analyses) or by enrichment approaches (such as immunopurifications). Moreover, this approach corresponds to a cost-effective and simple normalization method altrenative, therefore being an appealing strategy for large quantitative screening, as the analysis of clinical cohorts for biomarker discovery.

SWATH-MS as a tool for biomarker discovery: From basic research to clinical applications.

In the era of quantitative proteomics, where mass spectrometry plays a pivotal role, in particular associated with the use of data-independent acquisition, it is time to perform an overview of this growing field with special focus on one of the most promising approaches: SWATH-MS, and to present future perspectives for its application as a translational tool. Therefore, a summary of this technique is presented focusing on two key relevant concepts associated with its application in biomarker discovery: the protein library and the individual digital maps concepts. It is also the purpose of this review to document the likely impact of SWATH-MS in both fundamental and translational research including biomarker identification and creation of diagnostic tools. To that end, the two concepts referred above were integrated with ongoing technical developments. Finally, some of the current restrictions for the implementation of SWATH-MS on a large scale are identified, and potential solutions presented, namely protocol standardization combined with the use of the proper standards.

A reference library of peripheral blood mononuclear cells for SWATH-MS analysis.

PURPOSE: Peripheral blood mononuclear cells (PBMCs) play quite diverse and important roles in monitoring immune homeostasis. Thus, this subset of blood cells may provide access to potential physiological relevant biomolecules, namely proteins. For this reason, PBMCs represent a promising alternative biological sample in scientific research, particularly as a source of potential biomarkers. Prior proteomic studies of PBMCs from healthy individuals focused only on a qualitative analysis, lacking the quantitative analysis information crucial for biomarker discovery, since most of the biological alterations result in slight changes in protein levels, not affecting the overall proteome composition. Therefore, this study aimed to provide a comprehensive PBMCs proteome library to be use in protein quantification by SWATH-MS. EXPERIMENTAL DESIGN: A SWATH-MS library was generated by a comprehensive 2D-LC-MS/MC analysis of a pooled sample of PBMCs from 6 different donors. GeLC-SWATH-MS analysis of the 6 donors was further used to test the generated library. RESULTS: The generated library comprises 1102 proteins involved in diverse human diseases and in immune system-related pathways. When tested in biological samples this library allowed the quantification of 920 different proteins, and around 700 per individual. CONCLUSIONS AND CLINICAL RELEVANCE: The provided PBMCs proteome library will be useful in further studies that aim to reproducibly quantify a large number of PBMCs' proteins without the need to perform protein identification. Furthermore, this robust microLC-SWATH-MS analysis is suitable with clinical practice.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome?

INTRODUCTION: The use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear. METHODS: In the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells. RESULTS: The present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome. CONCLUSIONS: Our results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications.

Short GeLC-SWATH: a fast and reliable quantitative approach for proteomic screenings.

The quantification of large proteomes across multiple samples has become the major focus of proteomics. In addition to the advantages of in-gel digestion, the extensive time and sample handling required have precluded the use of this type of method for large quantitative screens. Therefore, an adaptation of the in-gel digestion method, termed short-GeLC, is proposed as a faster and more reproducible sample preparation method for quantitative approaches. The proposed methodology was compared with two well-established procedures for sample preparation, GeLC-MS and the classic liquid digestion followed by LC-MS, using a membrane protein-enriched sample. The results show that the short-GeLC approach substantially reduces the amount of sample handling and the overall time required for analysis compared with the gel-based methods without compromising the overall results at the protein identification level. Furthermore, the short-GeLC approach in combination with the SWATH acquisition method leads to the best quantitative results: more proteins were quantified, and the reproducibility was improved. Finally, this method performed well even on challenging samples enriched in membrane proteins.