Altered Protein Profile Research Articles

Comparative Proteomic Study of Retinal Ganglion Cells Undergoing Various Types of Cellular Stressors.

Retinal ganglion cell (RGC) damage serves as a key indicator of various retinal degenerative diseases, including diabetic retinopathy (DR), glaucoma, retinal arterial and retinal vein occlusions, as well as inflammatory and traumatic optic neuropathies. Despite the growing body of data on the RGC proteomics associated with these conditions, there has been no dedicated study conducted to compare the molecular signaling pathways involved in the mechanism of neuronal cell death. Therefore, we launched the study using two different insults leading to RGC death: glutamate excitotoxicity and optic nerve crush (ONC). C57BL/6 mice were used for the study and underwent NMDA- and ONC-induced damages. Twenty-four hours after ONC and 1 hour after NMDA injection, we collected RGCs using CD90.2 coupled magnetic beads, prepared protein extracts, and employed LC-MS for the global proteomic analysis of RGCs. Statistically significant changes in proteins were analyzed using the Shiny Go program to identify GO biological processes and molecular functions resulting from the treatment. We identified unique and common alterations in protein profiles in RGCs undergoing different types of cellular stressors. Additionally, we observed the absence of certain proteins in treated RGCs compared to the control group. Our study not only identified both unique and shared proteomic changes but also laid the groundwork for the future development of a therapeutic platform for testing gene candidates for DR and glaucoma.

Comparative proteomic study of retinal ganglion cells undergoing various types of cellular stressors

Retinal ganglion cell (RGC) damage serves as a key indicator of various retinal degenerative diseases, including diabetic retinopathy (DR), glaucoma, retinal arterial and retinal vein occlusions, as well as inflammatory and traumatic optic neuropathies. Despite the growing body of data on the RGC proteomics associated with these conditions, there has been no dedicated study conducted to compare the molecular signaling pathways involved in the mechanism of neuronal cell death. Therefore, we launched the study using two different insults leading to RGC death: glutamate excitotoxicity and optic nerve crush (ONC). C57BL/6 mice were used for the study and underwent NMDA- and ONC-induced damage. Twenty-four hours after ONC and 1 h after NMDA injection, we collected RGCs using CD90.2 coupled magnetic beads, prepared protein extracts, and employed LC-MS for the global proteomic analysis of RGCs. Statistically significant changes in proteins were analyzed to identify changes to cellular signaling resulting from the treatment. We identified unique and common alterations in protein profiles in RGCs undergoing different types of cellular stresses. Our study not only identified both unique and shared proteomic changes but also laid the groundwork for the future development of a therapeutic platform for testing gene candidates for DR and glaucoma.

Effects of nano freezing-thawing on myofibrillar protein of Atlantic salmon fillets: Protein structure and label-free proteomics

This study investigated the impact of magnetic nanoparticles (MNPs) -assisted cryogenic freezing integrated with MNPs combined microwave thawing (NNMT) on the structural integrity of myofibrillar proteins and alterations in protein profiles in salmon fillets. The NNMT showed the lowest myofibrillar fragmentation index (MFI) value (2.73 ± 0.31) among the four freezing-thawing groups. The myofibrillar structure exhibited the highest level of integrity, while the myofibrillar proteins demonstrated minimal aggregation and displayed the most stable secondary and tertiary structures in response to NNMT treatment. Compared with the other three treatments, NNMT exhibited a high abundance of ionic and hydrogen bonds, resulting in stronger interactions between the proteins and water molecules. The label-free proteomics analysis revealed that different freezing-thawing methods primarily affected the cytoskeletal proteins, with collagen and myosin being down-regulated due to degradation caused by cold stress and recrystallization. Additionally, NNMT demonstrated a superior capability in stabilizing salmon cytoskeletal proteins.

Effect of cadmium and lead on the morphology and protein profile of Calligonum comosum seeds

BackgroundSoils contaminated with heavy metals (HMs) pose a risk to human health via the food chain, as many edible plants absorb these metals. In turn, some of these plants could be used as phytoremediators for such soils. Calligonum comosum is an indigenous medicinal shrub that grows naturally in wide swaths of sandy soil in Saudi Arabia and has many advantages that render it a promising candidate for the treatment of HM-contaminated areas. But the impact of HM on this plant remains unknown, especially in the early stages of its development.ObjectiveThis investigation aimed to study the effects of lead (Pb) and cadmium (Cd) on the germination of C. comosum seeds under laboratory conditions, and assess the seeds’ response to these metals.MethodsThe C. comosum seed germination was monitored in Petri dishes containing Pb and Cd at increasing concentrations (25, 50, 75, and 100 µM) for up to 3 weeks. SDS-PAGE was used to examine the protein profile of germinated seeds and the western blot was used to assess the influence of HMs on the activities of the catalase enzyme and the beta subunit of ATP synthase (AtpB).ResultsThe germination rate and speed of C. comosum seeds were delayed by increasing concentrations of either Pb or Cd, but this effect was dose-dependent. SDS-PAGE analysis results revealed that exposure to both metals led to altered protein profiles as indicated by the resulting band intensities and disappearance of some proteins compared with the untreated controls. Further, the western blot analysis detected greater activity of catalase enzyme as well as AtpB in the Cd- and Pb-treated seeds.ConclusionC. comosum seeds treated with Cd or Pb enhance protein degradation and denaturation beside oxidative stress, leading to reduced seed viability. These results suggest oxidoreduction proteins and those involved in ATP synthesis are enhanced in C. comosum seeds in response to Cd and Pb stressors, which is a probable mechanism by which seeds may tolerate heavy metal stress.

Cross-sectional network analysis of plasma proteins/metabolites correlated with pathogenesis and therapeutic response in acute promyelocytic leukemia.

The treatment of PML/RARA+ acute promyelocytic leukemia (APL) with all-trans-retinoic acid and arsenic trioxide (ATRA/ATO) has been recognized as a model for translational medicine research. Though an altered microenvironment is a general cancer hallmark, how APL blasts shape their plasma composition is poorly understood. Here, we reported a cross-sectional correlation network to interpret multilayered datasets on clinical parameters, proteomes, and metabolomes of paired plasma samples from patients with APL before or after ATRA/ATO induction therapy. Our study revealed the two prominent features of the APL plasma, suggesting a possible involvement of APL blasts in modulating plasma composition. One was characterized by altered secretory protein and metabolite profiles correlating with heightened proliferation and energy consumption in APL blasts, and the other featured APL plasma-enriched proteins or enzymes catalyzing plasma-altered metabolites that were potential trans-regulatory targets of PML/RARA. Furthermore, results indicated heightened interferon-gamma signaling characterizing a tumor-suppressing function of the immune system at the first hematological complete remission stage, which likely resulted from therapy-induced cell death or senescence and ensuing supraphysiological levels of intracellular proteins. Overall, our work sheds new light on the pathophysiology and treatment of APL and provides an information-rich reference data cohort for the exploratory and translational study of leukemia microenvironment.

Significant alteration of protein profiles in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder, affecting women of child-bearing age, and the incidence rate is growing and assuming epidemic proportions. The etiology of PCOS remains unknown and there is no cure. Some animal models for PCOS have been established which have enhanced our understanding of the underlying mechanisms, but omics data for revealing PCOS pathogenesis and for drug discovery are still lacking. In the present study, proteomics analysis was used to construct a protein profile of the ovaries in a PCOS mouse model. The result showed a clear difference in protein profile between the PCOS and control group, with 495 upregulated proteins and 404 downregulated proteins in the PCOS group. The GO term and KEGG pathway analyses of differentially expressed proteins mainly showed involvement in lipid metabolism, oxidative stress, and immune response, which are consistent with pathological characteristics of PCOS in terms of abnormal metabolism, endocrine disorders, chronic inflammation and imbalance between oxidant and antioxidant levels. Also, we found that inflammatory responses were activated in the PCOS ovarium, while lipid biosynthetic process peroxisome, and bile secretion were inhibited. In addition, we found some alteration in unexpected pathways, such as glyoxylate and dicarboxylate metabolism, which should be investigated. The present study makes an important contribution to the current lack of PCOS ovarian proteomic data and provides an important reference for research and development of effective drugs and treatments for PCOS.

Environmental pro-oxidants induce altered envelope protein profiles in human keratinocytes.

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Mumefural Improves Recognition Memory and Alters ERK-CREB-BDNF Signaling in a Mouse Model of Chronic Cerebral Hypoperfusion.

Cognitive impairment resulting from chronic cerebral hypoperfusion (CCH) is known as vascular dementia (VaD) and is associated with cerebral atrophy and cholinergic deficiencies. Mumefural (MF), a bioactive compound found in a heated fruit of Prunus mume Sieb. et Zucc, was recently found to improve cognitive impairment in a rat CCH model. However, additional evidence is necessary to validate the efficacy of MF administration for treating VaD. Therefore, we evaluated MF effects in a mouse CCH model using unilateral common carotid artery occlusion (UCCAO). Mice were subjected to UCCAO or sham surgery and orally treated with MF daily for 8 weeks. Behavioral tests were used to investigate cognitive function and locomotor activity. Changes in body and brain weights were measured, and levels of hippocampal proteins (brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase (ERK), cyclic AMP-response element-binding protein (CREB), and acetylcholinesterase (AChE)) were assessed. Additionally, proteomic analysis was conducted to examine the alterations in protein profiles induced by MF treatment. Our study showed that MF administration significantly improved cognitive deficits. Brain atrophy was attenuated and MF treatment reversed the increase in AChE levels. Furthermore, MF significantly upregulated p-ERK/ERK, p-CREB/CREB, and BDNF levels after UCCAO. Thus, MF treatment ameliorates CCH-induced cognitive impairment by regulating ERK/CREB/BDNF signaling, suggesting that MF is a therapeutic candidate for treating CCH.

Small extracellular vesicles derived from four dimensional-culture of mesenchymal stem cells induce alternatively activated macrophages by upregulating IGFBP2/EGFR to attenuate inflammation in the spinal cord injury of rats.

Effectively reducing the inflammatory response after spinal cord injury (SCI) is a challenging clinical problem and the subject of active investigation. This study employed a porous scaffold-based three dimensional long-term culture technique to obtain human umbilical cord mesenchymal stem cell (hUC-MSC)-derived Small Extracellular Vesicles (sEVs) (three dimensional culture over time, the "4D-sEVs"). Moreover, the vesicle size, number, and inner protein concentrations of the MSC 4D-sEVs contained altered protein profiles compared with those derived from 2D culture conditions. A proteomics analysis suggested broad changes, especially significant upregulation of Epidermal Growth Factors Receptor (EGFR) and Insulin-like Growth Factor Binding Protein 2 (IGFBP2) in 4D-sEVs compared with 2D-sEVs. The endocytosis of 4D-sEVs allowed for the binding of EGFR and IGFBP2, leading to downstream STAT3 phosphorylation and IL-10 secretion and effective induction of macrophages/microglia polarization from the pro-inflammatory M1 to anti-inflammatory M2 phenotype, both in vitro and in the injured areas of rats with compressive/contusive SCI. The reduction in neuroinflammation after 4D-sEVs delivery to the injury site epicenter led to significant neuroprotection, as evidenced by the number of surviving spinal neurons. Therefore, applying this novel 4D culture-derived Small Extracellular Vesicles could effectively curb the inflammatory response and increase tissue repair after SCI.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis inadopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Foodomics reveals altered lipid and protein profiles of Antarctic krill (Euphausia superba) under different processing

Drying processing is an indispensable procedure for the application of krill. Different drying methods usually offer significant influence on the physicochemical properties of foodstuff. This study investigated the effects of freeze and hot-air drying approaches on krill using lipidomics and proteomics. Multivariate analysis indicated the dramatic differences between freeze dried and hot-air dried krill. A total of 276 lipids were observed to be changed, including phosphatidylcholines (PC), triacylglycerols (TG) as well as fatty acids (FA) and their conjugates. Interestingly, several long-chain polyunsaturated phospholipids, such as PC (20:5_22:6), PC (20:5_20:5) and PE (16:0_22:6) displayed higher contents in hot-air dried krill, suggesting that method was beneficial to the high-quality krill oil extraction. On the other hand, the levels of 13 proteins in hot-air dried krill were noted to be higher, whereas other 101 proteins were found to be higher in freeze dried krill. These results indicated that hot-air drying processing reserved more phospholipids in krill oil, but degraded lots of krill tissue proteins. The freeze drying maintained more proteins in tissue, increasing the risk of deterioration for long-term storge. Taken together, this comprehensive analysis provided novel insights into the processing of krill to facilitate the utilization of krill resource.

Proteome profiling of the prefrontal cortex of Fmr1 knockout mouse reveals enhancement of complement and coagulation cascades

Fragile X mental retardation protein (FMRP) deficit resulted from mutations in its encoded fragile X mental retardation 1 (Fmr1) gene is a common inherited cause of Fragile X syndrome (FXS) characterized by intellectual disability and autism spectrum disorder (ASD). The FMRP absence-induced altered gene expression in prefrontal cortex (PFC) are associated with autistic behaviors. However, there lacks a large-scale protein profiling in the PFC upon loss of FMRP. This study used a TMT-labeled proteomic analysis to identify a protein profile of the PFC in the Fmr1 knockout mouse. A total of 5886 proteins were identified in the PFC with 100 differentially abundant proteins (DAPs) in response to FMRP deficiency. Bioinformatical analyses showed that these DAPs were mostly enriched in immune system, extracellular part and complement and coagulation cascades. The complement and coagulation cascades include 6 upregulated proteins (SERPING1, C1QA, C3, FGA, FGB and FGG), which are associated with fibrin degradation, cell lysis, degranulation chemotaxis and phagocytosis linked to activation of immune and inflammatory responses. Thus, our data provide an altered protein profile upon loss of FMRP in the PFC, and suggest that the enhancement of complement and coagulation cascades might contribute to etiological and pathogenic roles of ASD in FXS. SignificanceThe etiology of autism spectrum disorder (ASD), a group of neurobiological disorders characterized by deficits in social interaction barriers and other abnormal behaviors, is still elusive. Autistic-like phenotypes are present in both Fragile X syndrome (FXS) patients and FMRP-deficiency FXS models. Given that prefrontal cortex is a critical brain area for social interaction, the FMRP absence induced-changes of a subset of proteins might contribute to ASD in FXS. Using a comprehensive proteomic analysis, this study provides a prefrontal protein profile of the FMRP-absent mouse with a total of 100 differentially abundant proteins (DAPs). Bioinformatic analyses suggest that these DAPs are mainly involved in the regulations of immune system and complement and coagulation cascades. We also show that 6 upregulated proteins (SERPING1, C1QA, C3, FGA, FGB and FGG) in the complement and coagulation cascades are associated with fibrin degradation, cell lysis, degranulation chemotaxis and phagocytosis regarding dysregulation of immune and inflammatory responses in the prefrontal cortex. Therefore, this study suggests that these FMRP-deficient DAPs in the prefrontal cortex might contribute to the etiology and pathogenesis of ASD in FXS.

Response to microplastic exposure: An exploration into the sea urchin immune cell proteome

It is now known that the Mediterranean Sea currently is one of the major hotspot for microplastics (MPs; < 5 mm) pollution and that the risks will be even more pronounced in the coming years. Thus, the in-depth study of the mechanisms underlying the MPs toxicity in key Mediterranean organisms, subjected to high anthropic pressures, has become a categorical imperative to pursue. Here, we explore for the first time the sea urchins immune cells profile combined to their proteome upon in vivo exposure (72 h) to different concentrations of polystyrene-microbeads (micro-PS) starting from relevant environmental concentrations (10, 50, 103, 104 MP/L). Every 24 h, immunological parameters were monitored. After 72 h, the abundance of MPs was examined in various organs and coelomocytes were collected for proteomic analysis based on a shotgun label free proteomic approach. While sea urchins treated with the lowest concentration tested (10 and 50 micro-PS/L) did not show the presence of micro-PS in any tissue, in the specimens exposed to the highest concentration (103 and 104 micro-PS) there was an internalisation of 9.75 ± 2.75 and 113.75 ± 34.5 MP/g, respectively. Proteomic analyses revealed that MPs exposure altered coelomocytes protein profile not only compared to the control group but also among the different micro-PS concentrations and these variations are micro-PS concentration dependent. The proteins exclusively expressed in the coelomocytes of specimens exposed to MPs are mainly metabolite interconversion enzymes, involved in cellular processes, indicating a severe alteration of the cellular metabolic pathways. Overall, these findings provide new insights on the mode of action of MPs in the sea urchin immune cells both at the molecular and cellular level.

Plasma protein profiling analysis in patients with atrial fibrillation before and after three different ablation techniques.

There are controversies on the pathophysiological alteration in patients with atrial fibrillation (AF) undergoing pulmonary vein isolation using different energy sources. We evaluated the changes in plasma proteins in acute phase post-ablation in patients receiving cryoballoon ablation, radiofrequency balloon ablation, or radiofrequency ablation. Blood samples from eight healthy controls and 24 patients with AF were taken on the day of admission, day 1, and day 2 post-ablation and analyzed by the Olink proximity extension assay. Proteins were identified and performed with enrichment analysis. Protein-protein interaction network and module analysis were conducted using Cytoscape software. Of 181 proteins, 42 proteins in the cryoballoon group, 46 proteins in the radiofrequency balloon group, and 43 proteins in the radiofrequency group significantly changed after ablation. Most of the proteins altered significantly on the first day after ablation. Altered proteins were mainly involved in cytokine-cytokine receptor interaction. Both balloon-based ablations showed a similar shift toward enhancing cell communication and regulation of signaling while inhibiting neutrophil chemotaxis. However, radiofrequency ablation presented a different trend. Seed proteins, including osteopontin, interleukin-6, interleukin-10, C-C motif ligand 8, and matrix metalloproteinase-1, were identified. More significant proteins associated with hemorrhage and coagulation were selected in balloon-based ablations by machine learning. Plasma protein response after three different ablations in patients with AF mainly occurred on the first day. Radiofrequency balloon ablation shared similar alteration in protein profile as cryoballoon ablation compared with radiofrequency ablation, suggesting that lesion size rather than energy source is the determinant in pathophysiological responses to the ablation.

Radiation-Induced Intestinal Normal Tissue Toxicity: Implications for Altered Proteome Profile

Radiation-induced toxicity to healthy/normal intestinal tissues, especially during radiotherapy, limits the radiation dose necessary to effectively eradicate tumors of the abdomen and pelvis. Although the pathogenesis of intestinal radiation toxicity is highly complex, understanding post-irradiation alterations in protein profiles can provide crucial insights that make radiotherapy safer and more efficient and allow for increasing the radiation dose during cancer treatment. Recent preclinical and clinical studies have advanced our current understanding of the molecular changes associated with radiation-induced intestinal damage by assessing changes in protein expression with mass spectrometry-based approaches and 2-dimensional difference gel electrophoresis. Studies by various groups have demonstrated that proteins that are involved in the inflammatory response, the apoptotic pathway, reactive oxygen species scavenging, and cell proliferation can be targeted to develop effective radiation countermeasures. Moreover, altered protein profiles serve as a crucial biomarkers for intestinal radiation damage. In this review, we present alterations in protein signatures following intestinal radiation damage as detected by proteomics approaches in preclinical and clinical models with the aim of providing a better understanding of how to accomplish intestinal protection against radiation damage.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Label-free based proteomics revealed the specific changes of muscle proteins in pike eel (Muraenesox cinereus) under cold stress.

Chemical- and liquid chromatography coupled with mass spectrometry (LC-MS) based proteomics strategies were executed to investigate the alterations of protein profiles in pike eel (Muraenesox cinereus) muscle during chilling (CPE) and frozen (FPE) storage. Chemical results indicated that springiness and myofibrillar protein (MP) content of muscle tissues decreased significantly during 6days of chilled and 120days of frozen storage. LC-MS-based proteomics analysis suggested that great alterations occurred in muscle proteins mainly induced by cold stress. The differentially abundant proteins (DAPs) with low abundances in CPE and FPE samples included the annexins, fibronectin, ribosomal proteins, T-complex proteins, tubulin beta chain, and histones, which were mostly associated with the membrane structural constituents, cytoskeleton, and binding functional proteins. Results of eukaryotic cluster of orthologous group (KOG) verified that these identified DAPs were mainly converged in the cytoskeleton function resulting from cold conditions, which in turn affected the physical structure and chemical performances of muscle tissues.

Abstract P5-12-07: Proteomic profiling of extracellular vesicles released from leptin-treated breast cancer cells: A potential role in cancer metabolism

Abstract Extracellular vesicles (EVs), nanosized particles enclosed within a phospholipid bilayer membrane, are recognized key determinants of cell-to-cell communication in breast cancer, able to modulate phenotypic behavior of recipient cells by transferring their molecular cargo. Recently, we identified the adipokine leptin, whose circulating levels are elevated in obesity, as an important regulator of EV biogenesis and release in mammary carcinoma cells. Here, we focused on the identification of a specific leptin-induced EV proteomic signature in attempt to find molecular effectors associated with breast cancer progression. To this end, we analyzed protein content of EVs, isolated by ultracentrifugation method from conditioned media of ER-α positive MCF-7 breast cancer cells grown in the presence of leptin. EVs were fully characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Immunoblotting. Proteomic analysis of EV cargo, obtained by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS), revealed an altered protein profile, identifying 274 up-regulated proteins (FC&amp;gt;1.5) in EVs from leptin-treated cells compared to EV from untreated ones. Gene set enrichment analysis (GSEA) was used to explore Gene Ontology categories enriched in the entire list of up-regulated proteins. We identified a number of significantly enriched biologic processes mainly involved in energy production and mitochondrial metabolism, including “generation of precursor metabolites and energy” (GO:0006091), “fatty acid metabolic process” (GO:0006631), “mitochondrial respiratory chain” (GO:0033108), “mitochondrial gene expression” (GO:0140053) and “mitochondrial transport” (GO:0006839). Expression of up-regulated proteins, in both cell and EV lysates, was validated by immunoblotting. Protein-protein interaction network analysis revealed CYC1 (Cytochrome c-1), NDUFV2 (NADH:ubiquinone oxidoreductase core subunit V2), and NDUFAB1 (NADH:ubiquinone oxidoreductase subunit AB1), important subunits of mitochondrial complexes and enhancer of mitochondrial metabolism, as the most interactive proteins among that up-regulated in EVs released by leptin-treated breast cancer cells. This suggests a role for leptin-treated EVs in enhancing oxidative phosphorylation in both epithelial breast cancer cells and their surrounding microenvironment. Collectively, our data indicate that the adipokine leptin could induce in breast cancer cells the release of EVs enriched in specific proteins mainly involved in mitochondrial metabolism. Since, mitochondrial function is essential for supplying energy to support breast cancer cell growth and progression, understanding the metabolic mechanisms mediated by EV released by breast cancer cells may provide important clues to develop novel therapeutic approaches for treatment of breast cancer especially in obesity setting. Citation Format: Luca Gelsomino, Giusi La Camera, Ines Barone, Salvatore Panza, Giovanna Morello, Amanda Caruso, Chiara Chiodo, Daniela Bonofiglio, Cinzia Giordano, Sebastiano Andò, Stefania Catalano. Proteomic profiling of extracellular vesicles released from leptin-treated breast cancer cells: A potential role in cancer metabolism [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-07.

Proximate composition and changes in muscle proteins of dried salted abo (Otolithes ruber)

Dried salted abo is a popular fishery product in San Miguel Bay (SMB), Philippines. However, the development of this food product has not been fully explored. In this study, the proximate composition and muscle protein profile of dried salted abo was investigated after confirming the molecular identification of abo found in SMB. Sequence analysis of 16S ribosomal RNA and cytochrome c oxidase subunit I genes suggested that abo in SMB could be Otolithes ruber. Abo fillets were brined in 4%, 8%, and 12% salt concentrations (w/w) and sun-dried for 6 and 12 hrs. In both 6 hrs and 12 hrs drying time, moisture content decreased in samples brined with 12% salt, suggesting that the salt concentration could be lowered between 8–12% with 6 hrs drying time. Additionally, the proteolytic profiling by SDS-PAGE analysis detected 17 bands in the myofibrillar protein fraction. Subsequent mass spectrometry identified nine proteins, of which the myosin heavy chain, actin, and keratin were degraded while the other proteins remained stable. Moreover, at 12% salt concentration, changes in staining intensities in several proteins were observed at both 6 hrs and 12 hrs drying time. These findings revealed that the applied salting and drying conditions led to the degradation of myosin heavy chain and polymerization of actin, in addition to an overall alteration of protein profile, which could affect the texture of dried abo product. Overall, this study demonstrated molecular evidence for species identification of abo for the first time. The information on proximate composition and changes in the protein profile of dried salted abo obtained from this study could help the local dried fish industry develop high-quality dried abo products with low salt content.

TMT-Based Quantitative Proteomics Reveals Cochlear Protein Profile Alterations in Mice with Noise-Induced Hearing Loss.

Noise-induced hearing loss (NIHL) is a global occupational disease affecting health. To date, genetic polymorphism studies on NIHL have been performed extensively. However, the proteomic profiles in the cochleae of mice suffering noise damage remain unclear. The goal of this current study was to perform a comprehensive investigation on characterizing protein expression changes in the cochlea based on a mouse model of NIHL using tandem mass tag (TMT)-labeling quantitative proteomics, and to reveal the potential biomarkers and pathogenesis of NIHL. Male C57BL/6J mice were exposed to noise at 120 dB SPL for 4 h to construct the NIHL mouse model. The levels of MDA and SOD, and the production of proinflammatory cytokines including TNF-α and IL-6 in the mice cochleae, were determined using chemical colorimetrical and ELISA kits. Moreover, differentially expressed proteins (DEPs) were validated using Western blotting. The mouse model showed that the ABR thresholds at frequencies of 4, 8, 12, 16, 24 and 32 kHz were significantly increased, and outer hair cells (HCs) showed a distinct loss in the noise-exposed mice. Proteomics analysis revealed that 221 DEPs were associated with NIHL. Bioinformatics analysis showed that a set of key inflammation and autophagy-related DEPs (ITGA1, KNG1, CFI, FGF1, AKT2 and ATG5) were enriched in PI3K/AKT, ECM-receptor interaction, and focal adhesion pathways. The results revealed that the MDA level was significantly increased, but the activity of SOD decreased in noise-exposed mice compared to the control mice. Moreover, TNF-α and IL-6 were significantly increased in the noise-exposed mice. Western blotting revealed that the expression levels of ITGA1, KNG1, and CFI were upregulated, but FGF1, AKT2, and ATG5 were significantly downregulated in noise-exposed mice. This study provides new scientific clues about the future biomarkers and pathogenesis studies underlying NIHL. Furthermore, the findings suggest that the validated DEPs may be valuable biomarkers of NIHL, and inflammation and autophagy may be pivotal mechanisms that underlie NIHL.