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20 pages, 5788 KiB

Open AccessArticle

Multi-Omics Characterization of Colon Mucosa and Submucosa/Wall from Crohn’s Disease Patients

by Liang Jin, Michael Macoritto, Jing Wang, Yingtao Bi, Fei Wang, Abel Suarez-Fueyo, Jesus Paez-Cortez, Chenqi Hu, Heather Knight, Ivan Mascanfroni, Matthew M. Staron, Annette Schwartz Sterman, Jean Marie Houghton, Susan Westmoreland and Yu Tian

Int. J. Mol. Sci. 2024, 25(10), 5108; https://doi.org/10.3390/ijms25105108 - 8 May 2024

Viewed by 1143

Abstract

Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by transmural disease. The concept of transmural healing (TH) has been proposed as an indicator of deep clinical remission of CD and as a predictor of favorable treatment endpoints. Understanding the [...] Read more.

Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by transmural disease. The concept of transmural healing (TH) has been proposed as an indicator of deep clinical remission of CD and as a predictor of favorable treatment endpoints. Understanding the pathophysiology involved in transmural disease is critical to achieving these endpoints. However, most studies have focused on the intestinal mucosa, overlooking the contribution of the intestinal wall in Crohn’s disease. Multi-omics approaches have provided new avenues for exploring the pathogenesis of Crohn’s disease and identifying potential biomarkers. We aimed to use transcriptomic and proteomic technologies to compare immune and mesenchymal cell profiles and pathways in the mucosal and submucosa/wall compartments to better understand chronic refractory disease elements to achieve transmural healing. The results revealed similarities and differences in gene and protein expression profiles, metabolic mechanisms, and immune and non-immune pathways between these two compartments. Additionally, the identification of protein isoforms highlights the complex molecular mechanisms underlying this disease, such as decreased RTN4 isoforms (RTN4B2 and RTN4C) in the submucosa/wall, which may be related to the dysregulation of enteric neural processes. These findings have the potential to inform the development of novel therapeutic strategies to achieve TH. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Figure 1
Histological analysis of the colon mucosa and submucosa/wall. (A). Human normal colon, Crohn’s disease non-inflamed, and CD inflamed histologic sections stained with CD45 (DAB) and Alcian blue (top row), EpCam (red), smooth muscle actin (DAB), and Alcian blue (middle row and bottom row). Nuclear fast red counter stain. MM = muscularis mucosae, SM = submucosa, IC = inner circular muscle layer, OL = outer longitudinal muscle layer; (B) Human CD inflamed with tertiary lymphoid organs (TLOs) stained with CD45 (DAB) (left column), CD3 (purple), CD19 (yellow) double IHC (middle), and pan-myeloid IBA (DAB) (right), CD40 (DAB) (bottom left). Hematoxylin counterstain. Fluorescent multiplex of TLO in CD CD19 (red), CD3 (green), IBA1 (yellow) (bottom right). Full article ">Figure 2
Principal component analysis (PCA) and differential expression (DE) analysis of transcriptomics and proteomics. (A). PCA plot of mucosa transcriptomics. (B). PCA plot of mucosa proteomics. (C). PCA plot of submucosa/wall transcriptomics. (D). PCA plot of submucosa/wall proteomics. The values following PC1/PC2 in PCA plots represent the percentage of variance explained by the respective principal component (PC). (E). Comparison of differentially expressed genes (DEGs) between commonly detected genes from transcriptomics of mucosa and submucosa/wall. (F). Comparison of differentially expressed proteins (DEPs) between commonly detected proteins from proteomics of mucosa and submucosa/wall. Dashed lines represent the adjusted p-value/false discovery rate (FDR) equal to 0.05. FDR < 0.05 was considered statistically significant. Full article ">Figure 3
Weighted gene co-expression network analysis (WGCNA) of transcriptomics. WGCNA was performed on transcriptomics data. Modules were identified that were significantly correlated with diseased (up) or non-IBD control tissue (down) in mucosa (A) and submucosa/wall (B). GOBP ORA was performed and a general biological role for each module was identified using the results. Three GO terms that best represent the overarching biology of each module are shown. All significant terms for each module are shown in <a href="#app1-ijms-25-05108" class="html-app">Supplemental Data S4 and S5</a>. Full article ">Figure 4
Weighted gene co-expression network analysis (WGCNA) of proteomics. WGCNA was performed on proteomics data. Modules were identified that were significantly correlated with diseased (up) or non-IBD control tissue (down) in mucosa (A) and submucosa/wall (B). GOBP ORA was performed and a general biological role for each module was identified using the results. Three GO terms that best represented the overarching biology of each module are shown. All significant terms for each module are shown in <a href="#app1-ijms-25-05108" class="html-app">Supplemental Data S6 and S7</a>. Full article ">Figure 5
Comparison of estimated cell fractions of immune cells in Crohn’s disease (CD). Cell fractions were estimated from transcriptomics using cell-type deconvolution. Boxplots represent the relative fractions of (A) T cells in mucosa, (B) T cells in submucosa/wall, (C) B cells in mucosa, and (D) B cells in submucosa/wall in CD inflamed (yellow), CD non-inflamed (grey), and non-IBD controls (blue). The p-values were calculated using the Wilcoxon rank sum test. A p-value < 0.05 was considered statistically significant, and “n.s.” indicates not significant. The dots represent all observations that are below the first quantile − 1.5 × interquartile range (IQR) or above the third quantile + 1.5 × IQR. Full article ">Figure 6
Comparison of estimated cell fractions of non-immune cells in Crohn’s disease (CD). Cell fractions were estimated from transcriptomics using cell-type deconvolution. Boxplots represent the relative fractions of (A) epithelial cells in mucosa, (B) fibroblasts in mucosa, (C) fibroblasts in submucosa/wall, (D) endothelial cells in mucosa, and (E) endothelial cells in submucosa/wall in CD inflamed (yellow), CD non-inflamed (grey), and non-IBD controls (blue). The p-values were calculated using the Wilcoxon rank sum test. A p-value < 0.05 was considered statistically significant, and “n.s.” indicates not significant. The dots represent all observations that are below the first quantile − 1.5 × interquartile range (IQR) or above the third quantile + 1.5 × IQR. Full article ">Figure 7
Identification of protein isoforms from mucosa and submucosa/wall. Violin plots comparing the protein and mRNA isoform abundances of (A) FBLN1, and (B) RTN4 between mucosa and submucosa/wall. Asterisks denote a p-value < 0.05 from the Wilcoxon rank sum test. CD inflamed, CD non-inflamed, and non-IBD control samples are represented by yellow, red, and cyan, respectively. Full article ">

23 pages, 2814 KiB

Open AccessArticle

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations

by Frode Selheim, Elise Aasebø, Håkon Reikvam, Øystein Bruserud and Maria Hernandez-Valladares

Int. J. Mol. Sci. 2024, 25(10), 5080; https://doi.org/10.3390/ijms25105080 - 7 May 2024

Cited by 1 | Viewed by 899

Abstract

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased [...] Read more.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Comparison of the AML cell proteome of FAB-M4/M5 patients with and without NPM1 insertions (-Ins). (a,b) show significantly enriched Reactome pathways and protein–protein interaction (PPI) networks of differentially expressed proteins with significantly higher expression in NPM1-Ins patients; (c,d) show significantly enriched Reactome pathways and PPI clusters of regulated proteins with a higher expression for patients without NPM1-Ins than for NPM1-Ins FAB-M4/M5 patients. Significance of PPI cohesiveness is shown with p-values of a one-sided Mann–Whitney U test. Full article ">Figure 2
The AML cell phosphoproteome of FAB-M4/M5 patients. All analyses are based on those phosphosites that differed significantly between the two groups; the upper part (a–c) presents the results for protein sites with increased phosphorylation levels for patients with NPM1-Ins, and the lower part (d–f) presents the results for patients without NPM1-Ins. For both groups we show Reactome pathway analysis (a,d), PPI analysis (b,e), and sequence motif analysis of the ±5 amino acids flanking the differentially regulated phosphorylation sites (c,f). * Significance was only detected with unadjusted p-values < 0.05. Full article ">Figure 3
Unsupervised hierarchical clustering of FAB-M4/M5 patients with and without (referred to as NPM1-wt in the figure) NPM1-Ins. The analysis is based on the expression of COMM domain-containing proteins, GPI transamidase proteins, and vacuolar sorting proteins showing differential expression in ANOVA and, in addition, being included in PPI networks. Full article ">

17 pages, 2466 KiB

Open AccessArticle

Proteomic Analysis of Prehypertensive and Hypertensive Patients: Exploring the Role of the Actin Cytoskeleton

by Sarah Al Ashmar, Gulsen Guliz Anlar, Hubert Krzyslak, Laiche Djouhri, Layla Kamareddine, Shona Pedersen and Asad Zeidan

Int. J. Mol. Sci. 2024, 25(9), 4896; https://doi.org/10.3390/ijms25094896 - 30 Apr 2024

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Abstract

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying [...] Read more.

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤−1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Proteomic analysis of the groups. (A) Orthogonal partial least squares discriminant analysis (OPLS-DA) scatter plot showing the separation between the three groups based on their proteomic signature. (B) Heatmap of all 1305 proteins showing an unsupervised hierarchical clustering of altered proteins between the three groups. PC1: principal component 1; PC2: principal component. Full article ">Figure 2
Volcano plots. (A) Volcano plot representing differentially expressed proteins between control and hypertension groups. (B) Volcano plot representing differentially expressed proteins between control and prehypertension groups. (C) Volcano plot representing differentially expressed proteins between hypertension and prehypertension groups. Full article ">Figure 3
(A) KEGG pathway analysis of the significant proteins resulting from ANOVA. Cluster 1 (enrichment score = 12.27) is represented in a pie chart in terms of the number of proteins mapped per pathway. (B) Network analysis of the significant proteins between prehypertension and hypertension with AUC ≥ 0.8. Number of nodes: 25; number of edges: 54; average node degree: 4.32; average local clustering coefficient: 0.482; expected number of edges: 17; protein–protein interaction enrichment p-value: 1.15 × 10−12. Full article ">Figure 4
Boxplots and ROC curves of actin cytoskeleton-related proteins significantly different between prehypertension and hypertension groups. The relative fluorescence unit (RFU) was used for boxplots. AUC: Area Under Curve, CI: Confidence Interval; CAMK2B: Calcium/calmodulin-dependent protein kinase type II subunit beta; CAMK2D: Calcium/calmodulin-dependent protein kinase type II subunit delta; GSK-3 α/β: Glycogen synthase kinase-3 alpha/beta; GRB2: Growth factor receptor-bound protein 2; LYN: Tyrosine-protein kinase Lyn; LYNB: Tyrosine-protein kinase Lyn isoform B; VAV1: Proto-oncogene vav; RAC1: Ras-related C3 botulinum toxin substrate 1; CSK: Tyrosine-protein kinase CSK; TPM4: Tropomyosin alpha-4 chain; SRC: Proto-oncogene tyrosine-protein kinase Src; YWHAB: 14-3-3 protein beta/alpha; TEC: Tyrosine-protein kinase Tec; FER: Tyrosine-protein kinase Fer. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 5
ROC analysis of the actin cytoskeleton-related proteins significantly different between prehypertension and hypertension in the validation cohort. Full article ">

37 pages, 24151 KiB

Open AccessArticle

Nicotinamide Mononucleotide (NMN) Works in Type 2 Diabetes through Unexpected Effects in Adipose Tissue, Not by Mitochondrial Biogenesis

by Roua Gabriela Popescu, Anca Dinischiotu, Teodoru Soare, Ene Vlase and George Cătălin Marinescu

Int. J. Mol. Sci. 2024, 25(5), 2594; https://doi.org/10.3390/ijms25052594 - 23 Feb 2024

Viewed by 2394

Abstract

Nicotinamide mononucleotide (NMN) has emerged as a promising therapeutic intervention for age-related disorders, including type 2 diabetes. In this study, we confirmed the previously observed effects of NMN treatment on glucose uptake and investigated its underlying mechanisms in various tissues and cell lines. [...] Read more.

Nicotinamide mononucleotide (NMN) has emerged as a promising therapeutic intervention for age-related disorders, including type 2 diabetes. In this study, we confirmed the previously observed effects of NMN treatment on glucose uptake and investigated its underlying mechanisms in various tissues and cell lines. Through the most comprehensive proteomic analysis to date, we discovered a series of novel organ-specific effects responsible for glucose uptake as measured by the IPGTT: adipose tissue growing (suggested by increased protein synthesis and degradation and mTOR proliferation signaling upregulation). Notably, we observed the upregulation of thermogenic UCP1, promoting enhanced glucose conversion to heat in intermuscular adipose tissue while showing a surprising repressive effect on mitochondrial biogenesis in muscle and the brain. Additionally, liver and muscle cells displayed a unique response, characterized by spliceosome downregulation and concurrent upregulation of chaperones, proteasomes, and ribosomes, leading to mildly impaired and energy-inefficient protein synthesis machinery. Furthermore, our findings revealed remarkable metabolic rewiring in the brain. This involved increased production of ketone bodies, downregulation of mitochondrial OXPHOS and TCA cycle components, as well as the induction of well-known fasting-associated effects. Collectively, our data elucidate the multifaceted nature of NMN action, highlighting its organ-specific effects and their role in improving glucose uptake. These findings deepen our understanding of NMN’s therapeutic potential and pave the way for novel strategies in managing metabolic disorders. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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12 pages, 2013 KiB

Open AccessCommunication

Variation of the 2D Pattern of Brain Proteins in Mice Infected with Taenia crassiceps ORF Strain

by Mariana Díaz-Zaragoza, Ricardo Hernández-Ávila, Abraham Landa and Pedro Ostoa-Saloma

Int. J. Mol. Sci. 2024, 25(3), 1460; https://doi.org/10.3390/ijms25031460 - 25 Jan 2024

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Abstract

Some parasites are known to influence brain proteins or induce changes in the functioning of the nervous system. In this study, our objective is to demonstrate how the two-dimensional gel technique is valuable for detecting differences in protein expression and providing detailed information [...] Read more.

Some parasites are known to influence brain proteins or induce changes in the functioning of the nervous system. In this study, our objective is to demonstrate how the two-dimensional gel technique is valuable for detecting differences in protein expression and providing detailed information on changes in the brain proteome during a parasitic infection. Subsequently, we seek to understand how the parasitic infection affects the protein composition in the brain and how this may be related to changes in brain function. By analyzing de novo-expressed proteins at 2, 4, and 8 weeks post-infection compared to the brains of the control mice, we observed that proteins expressed at 2 weeks are primarily associated with neuroprotection or the initial response of the mouse brain to the infection. At 8 weeks, parasitic infection can induce oxidative stress in the brain, potentially activating signaling pathways related to the response to cellular damage. Proteins expressed at 8 weeks exhibit a pattern indicating that, as the host fails to balance the Neuro-Immuno-Endocrine network of the organism, the brain begins to undergo an apoptotic process and consequently experiences brain damage. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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17 pages, 4066 KiB

Open AccessArticle

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation

by Roman Sakson, Lars Beedgen, Patrick Bernhard, K. Merve Alp, Nicole Lübbehusen, Ralph Röth, Beate Niesler, Marcin Luzarowski, Olga Shevchuk, Matthias P. Mayer, Christian Thiel and Thomas Ruppert

Int. J. Mol. Sci. 2024, 25(2), 1191; https://doi.org/10.3390/ijms25021191 - 18 Jan 2024

Cited by 1 | Viewed by 1305

Abstract

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging [...] Read more.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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22 pages, 3713 KiB

Open AccessArticle

Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy

by Ángela Marrugal, Irene Ferrer, Álvaro Quintanal-Villalonga, Laura Ojeda, María Dolores Pastor, Ricardo García-Luján, Amancio Carnero, Luis Paz-Ares and Sonia Molina-Pinelo

Int. J. Mol. Sci. 2023, 24(18), 13830; https://doi.org/10.3390/ijms241813830 - 7 Sep 2023

Cited by 1 | Viewed by 1453

Abstract

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy [...] Read more.

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Figure 1
Characterization of the lung adenocarcinoma cell lines panel used. Study of HSP90 protein expression, other related heat shock proteins, and EGFR and EML4-ALK client proteins in the cell lines under study. EGFR = EGFR mutation, KRAS = KRAS mutation, ALK = EML4-ALK translocation carrier, TN = triple negative (EGFR, KRAS and wild-type ALK). Full article ">Figure 2
Evaluation of HSP90 inhibition in lung adenocarcinoma cell lines. (A) HCC827, (B) H1650, (C) H1975, (D) A549, (E) H358, (F) H2009, (G) H1437, (H) H1781, (I) CALU-3, (J) H3122 and (K) H2228 were subjected to IC80 concentration of 17-AAG, IPI-504, STA-9090 and AUY-922 for 10, 24 or 48 h before Western blot analysis to study expression of HSP90α, HSP70 and the corresponding EGFR, EML4-ALK or CDK4 client proteins. Each experiment was performed in triplicate. Western blots correspond to a representative image of the replicates. - = untreated with inhibitor; h = hours. Full article ">Figure 3
Effectiveness of HSP90 gene silencing in the panel of lung adenocarcinoma cell lines. Western blot analysis of the validity of gene silencing of (A) HSP90α and (B) HSP90β in the HCC827 cell line using siRNAs from the commercial company Origene. In this same cell line, we chose a 48-h incubation with 30 pmol of siRNA_HSP90α_1 and siRNA_HSP90β_3 as the optimal conditions for (C) combined silencing of HSP90. HSP90α and HSP90β expression for the established conditions was studied in the (D) H1975, (E) H1650, (F) H2009, (G) A549, (H) Calu-3, and (I) H1781 cell lines. Verification of conditions for optimal silencing of HSP90α and HSP90β in (J,K) H2228 and (L,M) H358. Verification of HSP90α and HSP90β protein reduction in response to treatment with siRNAs (Dharmacon) of the (N) H3122 and (O) H1437 cell lines. - = Untransfected; Control = non-specific interfering RNA; siRNA_HSP90α = interfering RNA against HSP90α; siRNA_HSP90β = interfering RNA against HSP90β. Full article ">Figure 4
iTRAQ design of experiments. 100 μg of proteins from the different conditions to be analyzed, for each cell line under study, were digested with trypsin and labeled with iTRAQ reagents. Each of the thirteen fractions obtained was analyzed by LC-MS/MS and the data were combined to make the corresponding protein identification and quantification. VER = VER-155008; siRNA_α + β = gene silencing of HSP90α and HSP90β. Full article ">Figure 5
Representation of the significantly altered expression of proteins after HSP90 inhibition in the different lung adenocarcinoma molecular subtypes. Volcano plot of differentially expressed proteins after HSP90 inhibition and their corresponding p-value for (A) EGFR mutation, (B) EML4-ALK translocation, (C) KRAS mutation and (D) triple-negative cell lines. The X-axis corresponds to the change in expression of overexpressed (positive values) and underexpressed (negative values) proteins. The Y-axis represents the p-value of the corresponding change in expression. Full article ">Figure 6
Venn diagram showing proteins that were significantly and specifically deregulated after HSP90 inhibition in the different lung adenocarcinoma molecular subgroups. EGFR mut = group of cell lines with EGFR mutation, EML4-ALK = group of cell lines with translocation in ALK, KRAS mut = group of cell lines with KRAS mutation, TN = group of triple-negative cell lines. Full article ">Figure 7
Classification of altered pathways after HSP90 inhibition in the different lung adenocarcinoma molecular subgroups. EGFR mut = group of cell lines with mutation in EGFR, EML4-ALK = group of cell lines with translocation in ALK, KRAS mut = group of cell lines with KRAS mutation, TN = group of triple-negative cell lines. Full article ">Figure 8
In silico validation of ADI1 and RRP1 as response biomarkers of HSP90 inhibition in lung adenocarcinoma (A–D). KM plot of ADI expression according to first progression of disease (A) and overall survival (B), together with relationship of RRP1 expression with first progression (C) or overall survival (D) of lung adenocarcinoma patients. Full article ">

24 pages, 5257 KiB

Open AccessArticle

Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration

by Jacky M. K. Kwong, Joseph Caprioli, Joanne C. Y. Lee, Yifan Song, Feng-Juan Yu, Jingfang Bian, Ying-Hon Sze, King-Kit Li, Chi-Wai Do, Chi-Ho To and Thomas Chuen Lam

Int. J. Mol. Sci. 2023, 24(15), 12109; https://doi.org/10.3390/ijms241512109 - 28 Jul 2023

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Abstract

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at [...] Read more.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATH^TM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Review

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37 pages, 1001 KiB

Open AccessReview

Circulating Proteins as Diagnostic Markers in Gastric Cancer

by Ombretta Repetto, Roberto Vettori, Agostino Steffan, Renato Cannizzaro and Valli De Re

Int. J. Mol. Sci. 2023, 24(23), 16931; https://doi.org/10.3390/ijms242316931 - 29 Nov 2023

Cited by 8 | Viewed by 1954

Abstract

Gastric cancer (GC) is a highly malignant disease affecting humans worldwide and has a poor prognosis. Most GC cases are detected at advanced stages due to the cancer lacking early detectable symptoms. Therefore, there is great interest in improving early diagnosis by implementing [...] Read more.

Gastric cancer (GC) is a highly malignant disease affecting humans worldwide and has a poor prognosis. Most GC cases are detected at advanced stages due to the cancer lacking early detectable symptoms. Therefore, there is great interest in improving early diagnosis by implementing targeted prevention strategies. Markers are necessary for early detection and to guide clinicians to the best personalized treatment. The current semi-invasive endoscopic methods to detect GC are invasive, costly, and time-consuming. Recent advances in proteomics technologies have enabled the screening of many samples and the detection of novel biomarkers and disease-related signature signaling networks. These biomarkers include circulating proteins from different fluids (e.g., plasma, serum, urine, and saliva) and extracellular vesicles. We review relevant published studies on circulating protein biomarkers in GC and detail their application as potential biomarkers for GC diagnosis. Identifying highly sensitive and highly specific diagnostic markers for GC may improve patient survival rates and contribute to advancing precision/personalized medicine. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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34 pages, 2500 KiB

Open AccessReview

Searching for Atherosclerosis Biomarkers by Proteomics: A Focus on Lesion Pathogenesis and Vulnerability

by Gabriele Nieddu, Marilena Formato and Antonio Junior Lepedda

Int. J. Mol. Sci. 2023, 24(20), 15175; https://doi.org/10.3390/ijms242015175 - 14 Oct 2023

Cited by 2 | Viewed by 1475

Abstract

Plaque rupture and thrombosis are the most important clinical complications in the pathogenesis of stroke, coronary arteries, and peripheral vascular diseases. The identification of early biomarkers of plaque presence and susceptibility to ulceration could be of primary importance in preventing such life-threatening events. [...] Read more.

Plaque rupture and thrombosis are the most important clinical complications in the pathogenesis of stroke, coronary arteries, and peripheral vascular diseases. The identification of early biomarkers of plaque presence and susceptibility to ulceration could be of primary importance in preventing such life-threatening events. With the improvement of proteomic tools, large-scale technologies have been proven valuable in attempting to unravel pathways of atherosclerotic degeneration and identifying new circulating markers to be utilized either as early diagnostic traits or as targets for new drug therapies. To address these issues, different matrices of human origin, such as vascular cells, arterial tissues, plasma, and urine, have been investigated. Besides, proteomics was also applied to experimental atherosclerosis in order to unveil significant insights into the mechanisms influencing atherogenesis. This narrative review provides an overview of the last twenty years of omics applications to the study of atherogenesis and lesion vulnerability, with particular emphasis on lipoproteomics and vascular tissue proteomics. Major issues of tissue analyses, such as plaque complexity, sampling, availability, choice of proper controls, and lipoproteins purification, will be raised, and future directions will be addressed. Full article

(This article belongs to the Special Issue Proteomics and Its Applications in Disease 2.0)

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Figure 1

Figure 1
Picture showing the contribution of major acute cardiovascular events such as IHD and stroke to global deaths in 2019 [<a href="#B10-ijms-24-15175" class="html-bibr">10</a>]. Blue: non-communicable diseases. Red: communicable, maternal, neonatal, and nutritional diseases. Green: injuries. Full article ">Figure 2
Overview of the main targets of proteomic studies searching for both mechanisms of atherogenesis and biomarkers of atherosclerotic lesion presence and progression. Dotted lines represent partially unexplored paths. LCMs, laser-captured microdissections. Modified from [<a href="#B43-ijms-24-15175" class="html-bibr">43</a>]. Full article ">Figure 3
Lipoprotein purification and quality assessment procedures workflow. (I). Isopycnic salt gradient ultracentrifugation. (a) Ultracentrifugation tube containing 0.9 mL of plasma sample combined with NaBr, overlaid with 2.1 mL of a 0.6% NaCl solution; (b) self-generated density gradient following ultracentrifugation at 541,000× g for 3 h at 4 °C in a TL-100 series ultracentrifuge equipped with a TLA-100 fixed-angle rotor (Beckman Coulter, Brea, CA, USA), showing the three main lipoprotein classes; (c) LDL on the top of the tube following a further flotation step at d = 1.063 g/mL. (II). Concentration and dialysis using Amicon Ultra-0.5 mL centrifugal filter units (10 KDa MWCO, Merck-Millipore, Darmstadt, Germany). (III). Quality assessment using sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). Representative mono-dimensional profiles of HDL (d), LDL (e), and VLDL (f) fractions obtained by SDS-PAGE in either 12% T (for HDL, under reducing conditions) or 6% T (for both LDL and VLDL, under non-reducing conditions) resolving gels (modified from Nieddu et al. [<a href="#B55-ijms-24-15175" class="html-bibr">55</a>]). Full article ">Figure 4
Gene Ontology (GO) analysis of the 21 proteins identified by Finamore et al. [<a href="#B56-ijms-24-15175" class="html-bibr">56</a>], not yet included in the “likely” LDL proteins list of the Davidson’s Lab database, but already reported to be associated with LDL by either Dashty et al. [<a href="#B68-ijms-24-15175" class="html-bibr">68</a>] or Bancells et al. [<a href="#B69-ijms-24-15175" class="html-bibr">69</a>]. Full article ">

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