Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks.

Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.

Carbonic Anhydrase XII Expression Is Modulated during Epithelial Mesenchymal Transition and Regulated through Protein Kinase C Signaling.

Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

A Hidden Human Proteome Signature Characterizes the Epithelial Mesenchymal Transition Program.

BACKGROUND: Molecular changes associated with the initiation of the epithelial to mesenchymal transition (EMT) program involve alterations of large proteome-based networks. The role of protein products mapping to non-coding genomic regions is still unexplored. OBJECTIVE: The goal of this study was the identification of an alternative protein signature in breast cancer cellular models with a distinct expression of EMT markers. METHODS: We profiled MCF-7 and MDA-MB-231 cells using liquid-chromatography mass/spectrometry (LCMS/ MS) and interrogated the OpenProt database to identify novel predicted isoforms and novel predicted proteins from alternative open reading frames (AltProts). RESULTS: Our analysis revealed an AltProt and isoform protein signature capable of classifying the two breast cancer cell lines. Among the most highly expressed alternative proteins, we observed proteins potentially associated with inflammation, metabolism and EMT. CONCLUSION: Here, we present an AltProts signature associated with EMT. Further studies will be needed to define their role in cancer progression.

The Cancer Microbiota: EMT and Inflammation as Shared Molecular Mechanisms Associated with Plasticity and Progression.

With the advent of novel molecular platforms for high-throughput/next-generation sequencing, the communities of commensal and pathogenic microorganisms that inhabit the human body have been defined in depth. In the last decade, the role of microbiota-host interactions in driving human cancer plasticity and malignant progression has been well documented. Germ-free preclinical models provided an invaluable tool to demonstrate that the human microbiota can confer susceptibility to various types of cancer and can also modulate the host response to therapeutic treatments. Of interest, besides the detrimental effects of dysbiosis on cancer etiopathogenesis, specific microorganisms have been shown to exert protective activities against cancer growth. This has strong clinical implications, as restoration of the physiologic microbiota is being rapidly implemented as a novel anticancer therapeutic strategy. Here, we reviewed past and recent literature depicting the role of microbiota-host interactions in modulating key molecular mechanisms that drive human cancer plasticity and lead to malignant progression. We analyzed microbiota-host interactions occurring in the gut as well as in other anatomic sites, such as oral and nasal cavities, lungs, breast, esophagus, stomach, reproductive tract, and skin. We revealed a common ground of biological alterations and pathways modulated by a dysbiotic microbiota and potentially involved in the control of cancer progression. The molecular mechanisms most frequently affected by the pathogenic microorganisms to induce malignant progression involve epithelial-mesenchymal transition- (EMT-) dependent barrier alterations and tumor-promoting inflammation. This evidence may pave the way to better stratify high-risk cancer patients based on unique microenvironmental/microbial signatures and to develop novel, personalized, biological therapies.

Distinct Protein Expression Networks are Activated in Microglia Cells after Stimulation with IFN-γ and IL-4.

Microglia cells are the primary immune population of the central nervous system with a role in the regulation of several physiological and pathological conditions. Upon appropriate stimulation, microglia cells can be polarized in a pro-inflammatory M1-like or anti-inflammatory M2-like status. Biological processes and pathways engaged in microglia polarization are starting to be elucidated. To help clarify this, we used a liquid chromatography-mass spectrometry (LC-MS/MS) label free approach to characterize the proteomic profile of human microglia cell line (CHME-5) stimulated with gamma-interferon (IFN-γ) and interleukin-4 (IL-4) to induce a M1 or M2 phenotype, respectively. Outside the classical M1/M2 polarization markers, the M1 status appears to center around the activation of a classical inflammatory response and through the activation of multiple signaling pathways. M2 polarization resulted in a different pattern of protein modulation related to RNA and cellular metabolic processes. Together, our findings provide information regarding the protein changes specific to M1 and M2 activation states, and potentially link the polarization of microglia cells to the acquisition of a specific proteomic profile.